Translational control of GCN4 expression is mediated by AUG codons present in the 5' leader of the transcript: the third or fourth AUG codon is needed for repression in non-starvation conditions; the first is required for derepression in starvation conditions. Positive (GCN) and negative (GCD) transacting factors modulate the interactions between these upstream AUG codons. We have made the following advances in our understanding of this translational control mechanism: (1) the regulatory functions of both the 5' proximal and 3' proximal AUG codons can be mimicked by heterologous upstream open-reading-frames (URFs), demonstrating a lack of strict sequence specificity for URF regulatory functions. However, placing UJRFl downstream from URFs 3-4 abolishes regulation, showing that important sequence differences exist between these elements and their 5'-3' order is critical. (2) Substitutions at URFl with sequences found at URF4 show that the coding region and sequences 3' to the stop codon distinguish the functions of URFs 1 and 4. These results suggest that ribosomes must first translate URFl and resume scanning to move beyond URFs 3-4 in derepressing conditions. (4) lacZ fusions to URFs 1, 3, and 4 are all efficiently translated when no upstream AUG codons are present. Moreover, URFs 1 and 2 have nearly the same weak inhibitory effect on translation of URF3-, URF4-, and GCN4-lacZ fusions, arguing against differential effects of URFs 1-2 on translation of URFs 3- 4 versus GCN4. (5) Immunoblotting with antisera raised against GCN3 and GCDl shows that these factors are expressed constitutively, supporting the notion that their functions are controlled by protein-protein interactions. (6) ggcd12 and gcd2- 1 mutations were shown to be alleles of the same gene that have allele-specific interactions with GCN3; the carboxyl-terminus of GCD2 was found to be homologous to GCN3. Based on these findings, it is likely that GCD2 contains two domains, one of which competes with GCN3. (7) Complementation mapping and DNA sequence analysis of GCN2 was completed; a mutation in a conserved lysine residue in the putative GCN2 protein kinase domain was shown to completely inactivate GCN2 positive regulatory function. (8) Mutations in the structural genes for two yeast eIF2 subunits (sui) behave like gcd mutations in causing derepressed GCN4 expression independent of the positive regulator GCN2.